Journal of Molecular Biology

Recent progress in cryo-EM research has ignited a revolution in biological macromolecule structure determination. Resolution is an essential parameter for quality assessment of a cryo-EM density map, and it is known that resolution varies in different regions of a map. Currently available methods for local resolution estimation require manual adjustment of parameters and in some cases necessitate acquisition or de novo generation of so-called "half maps". Here, we developed CryoRes, a deep-learning algorithm to estimate local resolution directly from a single final cryo-EM density map, specifically by learning resolution-aware patterns of density map voxels through supervised training on a large dataset comprising 1,174 experimental cryo-EM density maps...

The human interactome is composed of around half a million interactions according to recent estimations and it is only for a small fraction of those that three-dimensional structural information is available. Indeed, the structural coverage of the human interactome is very low and given the complexity and time-consuming requirements of solving protein structures this problem will remain for the foreseeable future. Structural models, or predictions, of protein complexes can provide valuable information when the experimentally determined 3D structures are not available...

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The discovery of diverse bacterial CRISPR-Cas systems has reignited interest in understanding bacterial defense pathways while yielding exciting new tools for genome editing. CRISPR-Cas systems are widely distributed in prokaryotes, found in 40% of bacteria and 90% of archaea, where they function as adaptive immune systems against bacterial viruses (phage) and other mobile genetic elements. In turn, phage have evolved inhibitors, called anti-CRISPR proteins, to prevent targeting. Type V CRISPR-Cas12 systems have emerged as a particularly exciting arena in this co-evolutionary arms race...

The structural investigation of intrinsically disordered proteins (IDPs) requires ensemble models describing the diversity of the conformational states of the molecule. Due to their probabilistic nature, there is a need for new paradigms that understand and treat IDPs from a purely statistical point of view, considering their conformational ensembles as well-defined probability distributions. In this work, we define a conformational ensemble as an ordered set of probability distributions and provide a suitable metric to detect differences between two given ensembles at the residue level, both locally and globally...

In the present investigation, we have identified the functional significance of the highly conserved miR-140 binding site on the Hepatitis E Virus (HEV) genome. Multiple sequence alignment of the viral genome sequences along with RNA folding prediction indicated that the putative miR-140 binding site has significant conservation for sequence and secondary RNA structure among HEV genotypes. Site-directed mutagenesis and reporter assays indicated that an intact sequence of the miR-140 binding site is essential for HEV translation...

Understanding the base pairing of an RNA sequence provides insight into its molecular structure. By mining suboptimal sampling data, RNAprofiling 1.0 identifies the dominant helices in low-energy secondary structures as features, organizes them into profiles which partition the Boltzmann sample, and highlights key similarities/differences among the most informative, i.e.selected, profiles in a graphical format. Version 2.0 enhances every step of this approach. First, the featured substructures are expanded from helices to stems...

Mirogabalin is a novel gabapentinoid drug with a hydrophobic bicyclo substituent on the γ-aminobutyric acid moiety that targets the voltage-gated calcium channel subunit α2 δ1. Here, to reveal the mirogabalin recognition mechanisms of α2 δ1, we present structures of recombinant human α2 δ1 with and without mirogabalin analyzed by cryo-electron microscopy. These structures show the binding of mirogabalin to the previously reported gabapentinoid binding site, which is the extracellular dCache_1 domain containing a conserved amino acid binding motif...

We present an updated version of the Predicting Protein-Protein Interactions (PrePPI) webserver which predicts PPIs on a proteome-wide scale. PrePPI combines structural and non-structural clues within a Bayesian framework to compute a likelihood ratio (LR) for essentially every possible pair of proteins in a proteome; the current database is for the human interactome. The structural modeling (SM) clue is derived from template-based modeling and its application on a proteome-wide scale is enabled by a unique scoring function used to evaluate a putative complex...

Knr4/Smi1 proteins are specific to the fungal kingdom and their deletion in the model yeast Saccharomyces cerevisiae and the human pathogen Candida albicans results in hypersensitivity to specific antifungal agents and a wide range of parietal stresses. In S. cerevisiae, Knr4 is located at the crossroads of several signalling pathways, including the conserved cell wall integrity and calcineurin pathways. Knr4 interacts genetically and physically with several protein members of those pathways. Its sequence suggests that it contains large intrinsically disordered regions...

The nuclear pore complex (NPC) is a giant protein assembly that penetrates the double layers of the nuclear membrane. The overall structure of the NPC has approximately eightfold symmetry and is formed by approximately 30 nucleoporins. The great size and complexity of the NPC have hindered the study of its structure for many years until recent breakthroughs were achieved by integrating the latest high-resolution cryo-electron microscopy (cryo-EM), the emerging artificial intelligence-based modeling and all other available structural information from crystallography and mass spectrometry...

The detection of structural chromosomal abnormalities (SCA) is crucial for diagnosis, prognosis and management of many genetic diseases and cancers. This detection, done by highly qualified medical experts, is tedious and time-consuming. We propose a highly performing and intelligent method to assist cytogeneticists to screen for SCA. Each chromosome is present in two copies that make up a pair of chromosomes. Usually, SCA are present in only one copy of the pair. Convolutional neural networks (CNN) with Siamese architecture are particularly relevant for evaluating similarities between two images, which is why we used this method to detect abnormalities between both chromosomes of a given pair...

Stress granules (SGs) are cytosolic RNA-protein aggregates assembled during stress-induced translation arrest. Virus infection, in general, modulates and blocks SG formation. We previously showed that the model dicistrovirus Cricket paralysis virus (CrPV) 1A protein blocks stress granule formation in insect cells, which is dependent on a specific arginine 146 residue. CrPV-1A also inhibits SG formation in mammalian cells suggesting that this insect viral protein may be acting on a fundamental process that regulates SG formation...

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) systems provide bacteria and archaea with an adaptive immune response against invasion by mobile genetic elements like phages, plasmids, and transposons. These systems have been repurposed as very powerful biotechnological tools for gene editing applications in both bacterial and eukaryotic systems. The discovery of natural off-switches for CRISPR-Cas systems, known as anti-CRISPR proteins, provided a mechanism for controlling CRISPR-Cas activity and opened avenues for the development of more precise editing tools...

The Zα domain of ADARp150 is critical for proper Z-RNA substrate binding and is a key factor in the type-I interferon response pathway. Two point-mutations in this domain (N173S and P193A), which cause neurodegenerative disorders, are linked to decreased A-to-I editing in disease models. To understand this phenomenon at the molecular level, we biophysically and structurally characterized these two mutated domains, revealing that they bind Z-RNA with a decreased affinity. Less efficient binding to Z-RNA can be explained by structural changes in beta-wing, part of the Z-RNA-protein interface, and alteration of conformational dynamics of the proteins...

The human ATP-binding cassette (ABC) transporter ABCA1 plays a critical role in lipid homeostasis as it extracts sterols and phospholipids from the plasma membrane for excretion to the extracellular apolipoprotein A-I and subsequent formation of high-density lipoprotein (HDL) particles. Deleterious mutations of ABCA1 lead to sterol accumulation and are associated with atherosclerosis, poor cardiovascular outcomes, cancer, and Alzheimer's disease. The mechanism by which ABCA1 drives lipid movement is poorly understood, and a unified platform to produce active ABCA1 protein for both functional and structural studies has been missing...

Numerous viruses infecting bacteria and archaea encode CRISPR-Cas system inhibitors, known as anti-CRISPR proteins (Acr). The Acrs typically are highly specific for particular CRISPR variants, resulting in remarkable sequence and structural diversity and complicating accurate prediction and identification of Acrs. In addition to their intrinsic interest for understanding the coevolution of defense and counter-defense systems in prokaryotes, Acrs could be natural, potent on-off switches for CRISPR-based biotechnological tools, so their discovery, characterization and application are of major importance...

The identification of amino acid substitutions that both enhance the stability and function of a protein is a key challenge in protein engineering. Technological advances have enabled assaying thousands of protein variants in a single high-throughput experiment, and more recent studies use such data in protein engineering. We present a Global Multi-Mutant Analysis (GMMA) that exploits the presence of multiply-substituted variants to identify individual amino acid substitutions that are beneficial for the stability and function across a large library of protein variants...

Macromolecules change their shape (conformation) in the process of carrying out their functions. The imaging by cryo-electron microscopy of rapidly-frozen, individual copies of macromolecules (single particles) is a powerful and general approach to understanding the motions and energy landscapes of macromolecules. Widely-used computational methods already allow the recovery of a few distinct conformations from heterogeneous single-particle samples, but the treatment of complex forms of heterogeneity such as the continuum of possible transitory states and flexible regions remains largely an open problem...

Human WASP and N-WASP are homologous proteins that require the binding of multiple regulators, including the acidic lipid PIP2 and the small GTPase Cdc42, to relieve autoinhibition before they can stimulate the initiation of actin polymerization. Autoinhibition involves intramolecular binding of the C-terminal acidic and central motifs to an upstream basic region and GTPase binding domain. Little is known about how a single intrinsically disordered protein, WASP or N-WASP, binds multiple regulators to achieve full activation...